半夏曲发酵机制及主要优势真菌的发酵功能研究

Fermentation mechanism of Pinelliae Rhizoma(Banxiaqu)and fermentation function of main dominant fungi

目的探讨半夏曲的自然固态发酵机制。方法采用免培养、纯培养相结合的方法研究半夏曲发酵真菌和细菌区系构成及主要代谢功能,针对分离到的主要优势真菌的纯培养菌株,分别采用碘量法及分光光度法检测其产淀粉酶及蛋白酶情况,采用显微镜观察菌株对草酸钙结晶的破坏情况。结果半夏曲发酵过程中检测到丝衣孢霉Byssochlamys、根霉Rhizopus、曲霉Aspergillus、米勒酵母Millerozyma、假单胞菌Pseudomonas、芽孢杆菌Bacillus等属微生物。半夏曲发酵过程中真菌数量大幅增殖,前期及末期的真菌多样性远远高于发酵旺盛期,发酵40~64 h期间的样品与其他样品的真菌区系构成存在极显著差异(P<0.01)。丝衣孢霉属是唯一参与了整个半夏曲发酵过程的优势真菌,对米勒酵母、根毛霉、曲霉的生长具有明显抑制作用。细菌的数量及多样性均低于真菌,且其数量在半夏曲发酵过程中没有明显增长。微生物群落功能预测(phylogenetic investigation of communities by reconstruction of unobserved states,PICRUS)分析表明半夏曲发酵过程中的真菌与氨基酸代谢、糖代谢及核苷酸合成代谢密切相关。真菌菌株发酵实验证实,丝衣孢霉属真菌与α-淀粉酶的生成及草酸钙针晶破损有关。结论细菌不是半夏曲发酵过程中的主要功能微生物,丝衣孢霉是半夏曲发酵过程中的主要优势功能微生物,与半夏曲的淀粉酶活性和与刺激性降低密切相关。

Objective To explore the natural solid-state fermentation mechanism of Pinelliae Rhizoma(Banxiaqu),Methods The composition and main metabolic functions of fermentation fungi and bacteria were studied by culture and uncultured methods,and the isolated strains of main dominant fungi were detected on the amylase and protease production by iodometry and spectrophotometry respectively and observed by microscope on the destruction of calcium oxalate crystal.Results Byssochlamys,Rhizopus,Aspergillus,Millerozyma,Pseudomonas and Bacillus were involved in the fermentation of Pinelliae Rhizoma.The number of fungi increased significantly during the fermentation,and the fungal diversity in the early and late stage was much higher than that in the vigorous fermentation period.The fungal flora in the fermentative matrix during 40 h to 64 h were significantly different from other samples(P<0.01).Byssochlamys was the only dominant fungi species in samples from all stages of the fermentation process of Pinelliae Rhizoma,and obviously inhibited the growth of Millerozyma,Rhizomucor and Aspergillus.The number and diversity of bacteria was lower than that of the fungi,and the number had not increased obviously.Picrust functional prediction analysis showed that fungi in Pinelliae Rhizoma fermentation were closely related to amino acid metabolism,glucose metabolism and nucleotide synthesis metabolism.Fermentation by fungi strains showed that the Byssochlamys,not bacteria,was related to producing ofα-mylase and the damage of calcium oxalate crystal.Conclusion Byssochlamys were the main dominant functional microorganisms in the fermentation of Pinelliae Rhizoma,which were mainly contributed to the amylase activity and lower irritation of Banxiaqu.